The Cell Nucleus Fundamentals B, 2024 PDF

The Cell Nucleus Fundamentals B, 2024 Michael Lea E-mail: [email protected] Telephone: 973-972-5345 CELL NUCLEUS - LECTURE OUTLINE Suggested reading: Alberts: Molecular Biology of the Cell: 5th ed. p. 26-27, 195-245, 704-712 D.H. Lin and A. Hoelz, Nuclear Comings and Goings, The Scientist 30 (12)...

The Cell Nucleus Fundamentals B, 2024 Michael Lea E-mail: [email protected] Telephone: 973-972-5345 CELL NUCLEUS - LECTURE OUTLINE Suggested reading: Alberts: Molecular Biology of the Cell: 5th ed. p. 26-27, 195-245, 704-712 D.H. Lin and A. Hoelz, Nuclear Comings and Goings, The Scientist 30 (12): 24-29, 2016. Nuclear structure Chromatin, nucleosomes, histones Nuclear pores, nuclear import and export Cross-sectional view of a typical cell nucleus Nucleosomes as seen in the electron microscope A. Isolated chromatin B. decondensed chromatin Fig. 4-22, p. 211, 5th ed) Friedrich Miescher, discoverer of DNA Albrecht Kossel, discoverer of histones Nucleosomes contain DNA wrapped around a protein core of 8 histone molecules. Fig.5-22. Page 185 Irregularities in the 30 nm fiber featuring non-histone chromosomal proteins B. Structure of the histone fold C. H2A and H2B “handshake interaction” The assembly of the histone octamer The assembly of the histone octamer The bending of DNA in a nucleosome Covalent modification of core histone tails Electron micrograph of the nuclear side of the nuclear envelope NULEOPORINS Nucleoporins are the main components of the nuclear pore complex. There are about 30 of these proteins and they facilitate movement of molecules across the nuclear pores. Nuclear import receptors GTP hydrolysis by Ran provides directionality for nuclear transport Model for how the binding of Ran-GTP might cause nuclear import receptors to release their cargo Control of nuclear import during T-cell activation. NF-AT = Nuclear factor of activated T cells Breakdown and reformation of the nuclear envelope during mitosis LAMINS Lamins are structural proteins in the interphase nucleus. In mitosis, lamin B assembles into a matrixlike network through a process that requires Ran-GTP. This is an additional function for the Ran system in spindle assembly.. MEMBRANELESS ORGANELLES OR COMPARTMENTS IN THE NUCLEUS Membraneless compartment Known functions Nucleolus Ribosome biogenesis Nuclear speckle Gene expression regulation, splicing factor storage, pre-mRNA processing Nuclear stress body Gene expression regulation upon stress Histone locus body Pre-mRNA processing Cajal body snRNP maturation regulation and trafficking PML nuclear body Transcriptional regulation, protein storage Paraspeckle Gene expression regulation References: Crabtree M. and Nott T. Cellular droplets. The Scientist, December, 2018, pages 28-35. Garber K. How cells marshal their contents. Science 362: 1347. 2018. PARASPECKLE FUNCTION When a cell is stressed, various triggers can cause it to increase the production of the lncRNA NEAT1, leading to the formation of more paraspeckles. These bodies can grow to up to 2 micrometers in length, changing from a spherical shape to oblong and sometimes branched structures. They trap various proteins and mRNAs, hindering their function and thereby affecting the cell’s continued response to stress. Reference: A. Fox; Twinkles in the nucleus.The Scientist, December, pages 30-39, 2019 CELL NUCLEUS - LECTURE OBJECTIVES At the end of this session you should be able to Describe the components of nuclear structure Explain the role of histones in nucleosome structure and the compaction of DNA Identify the components of nuclear pores Describe the significance of nuclear import signals Distinguish the role of Ran in nuclear import and export of proteins Contrast the phosphorylation state of NF-AT in the nucleus and cytosol Describe the significance of lamin phosphorylation in mitosis Appreciate that the nucleus may contain membraneless compartments THE CELL CYCLE AND CELL DIVISION Michael Lea 2024 CELL CYCLE and CELL DIVISION LECTURE OUTLINE Suggested reading: Alberts – Molecular Biology of the Cell: 5th ed. p. 1053-1114 Eukaryotic cell division Mitosis and the cell cycle Cyclins and cyclin dependent kinases Control of cell division MEIOSIS In contrast to mitosis in somatic cells, meiosis occurs in gonads and results in the formation of gametes. In the first stage, meiosis I, following DNA synthesis the homologous chromosomes each containing sister chromatids are separated, thereby reducing the number of chromosomes to 23. There is no DNA synthesis between meiosis I and II. In meiosis II, sister chromatids are separated resulting in haploid gametes with 23 chromosomes MEIOSIS Progression through the cell cycle depends on cyclin-dependent protein kinases Distinct Cdks associate with different cyclins to trigger the different events of the cell cycle. Fig. 18-13, p621 The Major Cyclins and Cdks of Vertebrates Cyclin-Cdk Complex G1-Cdk G1/S-Cdk S-Cdk M-Cdk Cyclin D E A B From Table 18-2, Essential Cell Biology 2 CDK Partner Cdk4, Cdk6 Cdk2 Cdk2, Cdk1 Cdk1 S-PHASE CYCLIN-CDK COMPLEXES INITIATE DNA REPLICATION ONCE PER CELL CYCLE Studies in yeast have shown that the Origin Recognition Complex (ORC) remains associated with replication origins throughout the cell cycle. In early G1, Cdc6 associates with ORC. Aided by Cdc6, Mcm ring complexes then assemble on the adjacent DNA, resulting in the formation of the prereplicative complex. The S-Cdk assists in origin firing which involves the assembly of DNA polymerase and other replication proteins. The S-Cdk also blocks rereplication by causing the dissociation of Cdc6 from origins, its degradation, and the export of all excess Mcm out of the nucleus. Cdc6 and Mcm cannot return to reset an ORC-containing origin for another round of DNA replication until M-Cdk has been inactivated at the end of mitosis. ACTIVATION OF M-PHASE CYCLIN-CDK COMPLEXES TRIGGERS ENTRY INTO MITOSIS Cdk1 associates with M-cyclin as the levels of M-cyclin gradually rise. The resulting M-Cdk complex is phosphorylated on an activating site by Cdk-activating kinase (CAK) and on a pair of inhibitory sites by Wee I kinase. The resulting inactive M-Cdk complex is then activated at the end of G2 by the phosphatase Cdc25. Cdc25 is further stimulated by active M-Cdk, resulting in positive feedback. This feedback is enhanced by the ability of M-Cdk to inhibit Wee I. CELL CYCLE and CELL DIVISION – LECTURE OBJECTIVES At the end of this session you should be able to Describe the phases of the eukaryotic cell cycle Understand the role of microtubules in mitosis Contrast the mechanisms of mitosis and meiosis Describe the factors controlling the cell cycle Identify check points in the cell cycle Appreciate the factors regulating cyclin-dependent kinases Describe the molecular components of the DNA replication process Appreciate the role of inhibitory factors in cell cycle progression APOPTOSIS 2024 Michael Lea APOPTOSIS - LECTURE OUTLINE Suggested reading: Alberts – Molecular Biology of the Cell, 5th ed, p. 1115-1129 (Chapter 18) Apoptosis and Necrosis Caspases Extrinsic and Intrinsic Pathways Survival Factors Ferroptosis and Pyroptosis Efferocytosis Autophagy APOPTOSIS versus NECROSIS Patterns of Death Cell size Plasma membrane Mitochondria Organelle shape Nuclei DNA fragmentation Cell degradation Energy APOPTOSIS NECROSIS Single cells Shrinkage Fragmentation Continuity preserved Blebbed Phosphatidyl serine on surface Contents released Contracted Chromatin fragmented Internucleosomal cleavage Phagocytosis Requires ATP Groups of neighboring cells Swelling Smoothing Early lysis Swelling Swelling, Disruption Membrane disruption Diffuse and random Inflammation Does not require ATP A. Necrosis B. Apoptosis C. Disposal of apoptotic cell RECEPTOR-MEDIATED APOPTOSIS Mediated by cytokines/cytokine receptors belonging to the TNF/TNF receptor family. Examples: Fas ligand is primarily expressed in immune cells (T cells and Natural Killer cells). It binds the receptor called Fas. TNF binds the TNF receptor. Apo2 ligand (or TRAIL) binds DR4 (death receptor 4) and DR5 Apo3 ligand binds DR3 Four steps in Fas ligand/Fas mediated apoptosis: 1. Fas ligand binds to Fas leading to trimerization of the receptor 2. Recruitment of cytoplasmic proteins to cytosolic regions of Fas. FADD (Fas associated protein with death domain) is required for recruiting procaspase 8 and procaspase 10. 3. Caspase activation 4. Active caspase 8 cleaves procaspase 3 resulting in active caspase 3. Apoptosis results. MITOCHONDRIAL-DEPENDENT APOPTOSIS Mitochondrial-dependent apoptosis can be triggered by several factors including oxidants, calcium ions or translocation to the mitochondrial outer membrane of proapoptotic proteins (Bax, tBid). There is release of cytochrome c from the mitochondrial intermembrane space. This release of cytochrome c can be blocked by Bcl2. The apoptosome is formed from Apaf-1, dATP, cytochrome c and caspase 9. Activation of procaspase 9 leads to the cleavage of procaspase 3 resulting in the downstream events of apoptosis. Necroptosis Necroptosis is a programmed form of necrosis, or inflammatory cell death. Conventionally, necrosis is associated with unprogrammed cell death resulting from cellular damage or infiltration by pathogens, in contrast to orderly, programmed cell death via apoptosis. The discovery of necroptosis showed that cells can execute necrosis in a programmed fashion and that apoptosis is not always the preferred form of cell death. Furthermore, the immunogenic nature of necroptosis favors its participation in certain circumstances, such as aiding in defence against pathogens by the immune system. Necroptosis is well defined as a viral defense mechanism, allowing the cell to undergo "cellular suicide" in a caspase-independent fashion in the presence of viral caspase inhibitors to restrict virus replication. The signaling pathway responsible for carrying out necroptosis is generally understood. TNFα leads to stimulation of its receptor TNFR1. TNFR1 binding protein TNFR-associated death protein TRADD and TNF receptor-associated factor 2 TRAF2 signals to RIPK1 which recruits RIPK3 forming the necrosome also named ripoptosome. Phosphorylation of MLKL by the ripoptosome drives oligomerization of MLKL, allowing MLKL to insert into and permeabilize plasma membranes and organelles. Integration of MLKL leads to the inflammatory phenotype and release of damage-associated molecular patterns (DAMPs), which elicit immune responses. Reference: Wikipedia STIMULATION OF CELL DIVISION AND CELL DEATH BY MITOGENS Mitogens stimulate cell division but abnormal proliferation signals can cause cell-cycle arrest or cell death in normal cells. The binding of mitogens to cell-surface receptors leads to activation of Ras and a MAP kinase cascade. One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes including the gene encoding cyclin D and a gene encoding a subunit of the SCF ubiquitin ligase. Myc functions as a heterodimer with a protein called Max. Excessive levels of Myc cause the activation of p19ARF which binds and inactivates Mdm2 and thereby causes increased levels of p53 which may cause either cell-cycle arrest or apoptosis. SURVIVAL FACTORS CAN SUPPRESS APOPTOSIS In mammalian cells, the binding of survival factors to cell-surface receptors leads to activation of various protein kinases including protein kinase B (PKB) also known as AKT. PKB (AKT) phosphorylates and inactivates the Bcl-2 family member Bad. When not phosphorylated, Bad promotes apoptosis by binding and inhibiting Bcl-2. Once phosphorylated, Bad dissociates, freeing Bcl2 to suppress apoptosis. PKB (AKT) also suppresses death by phosphorylating and thereby inhibiting gene regulatory proteins of the Forkhead family that stimulate the transcription of genes that encode proteins that promote apoptosis. Akt has many substrates FERROPTOSIS AND PYROPTOSIS Ferroptosis is a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides, and is genetically and biochemically distinct from other forms of regulated cell death such as apoptosis. Pyroptosis is a cytotoxic process that occurs in macrophages after limited proteolysis of gasdermin D. The generation of an N-terminally cleaved fragment then creates large oligomeric membrane pores and causes lytic cell death ANASTASIS There is evidence that even after caspase activation there may be a reversal of apoptosis in a process that has been termed anastasis. If anastasis occurs late in apoptosis, the surviving cells may have genetic defects that can lead to malignancy. Reference: C.Q. Choi, Back from the brink of death. The Scientist, page 3239, February, 2019. Efferocytosis. Apoptotic cells release ‘find-me’ signals that recruit macrophages or immature dendritic cells to initiate efferocytosis. Receptor–ligand interactions between apoptotic cells and efferocytes trigger actin-mediated cytoskeletal rearrangements that enable engulfment of the apoptotic cell. Efferocytosis culminates in the release of anti-inflammatory cytokines. Efferocytes recognize signals on different types of dying cells. Many forms of cell death have been described (apoptosis, autophagy, necrosis, necroptosis, ferroptosis, caspase-independent cell death (CICD) and pyroptosis) that provide unique characteristics and surface-exposed macromolecules. AUTOPHAGY The term autophagy implies self-eating. Autophagy is the use of cellular material to provide energy under conditions of nutritional deprivation. The best known mechanism of autophagy involves the formation of a membrane around a targeted region of the cell to form an autophagosome. The resultant vesicle then fuses with a lysosome and the contents are degraded. Microautophagy involves direct uptake of proteins by lysosomes and chaperone-mediated autophagy involves specific recognition by the hsc70 complex. Autophagy serves to remove damaged organelles and proteins. This aspect complicates the use of inhibition of autophagy as a treatment modality for cancer. APOPTOSIS – LECTURE OBJECTIVES At the end of this session you should be able to Contrast the features of apoptosis and necrosis Describe the regulation of caspases in apoptosis Distinguish the extrinsic and intrinsic pathways of apoptosis Identify factors that favor cell survival Explain how excessive growth stimulation can result in apoptosis Describe alternative mechanisms for programmed cell death Understand that loss of ATP or NAD can lead to necrosis Appreciate the benefits of apoptosis and autophagy